The CD11a Binding Site of Efalizumab in Psoriatic Skin Tissue as Analyzed by Multi-Epitope Ligand Cartography Robot Technology

Abstract

Efalizumab (Raptiva^TM) is an immunomodulating recombinant humanized IgG1 monoclonal antibody that binds to CD11a, the α-subunit of leukocyte function antigen-1 (LFA-1). By blocking the binding of LFA-1 to ICAM-1, efalizumab inhibits the adhesion of leukocytes to other cell types and interferes with the migration of T lymphocytes to sites of inflammation (including psoriatic skin plaques). Analysis of the response in patients treated with efalizumab to date shows that distinct groups of responders and nonresponders to the drug exist. It would therefore be of great practical value to be able to predict which patients are most likely to respond to treatment, by identifying key parameters in the mechanism of action of efalizumab. Detailed investigation and detection of multiple epitopes in microcompartments of skin tissue has until recently been restricted by the available technology. However, the newly developed technique of Multi-Epitope Ligand Cartography (MELC) robot technology combines proteomics and biomathematical tools to visualize protein networks at the cellular and subcellular levels in situ, and to decipher cell functions. The MELC technique, which is outlined in this paper, was used to help characterize the binding of efalizumab to affected and unaffected psoriatic skin as compared to normal control skin under ex vivomodel conditions. Efalizumab was labeled with fluorescein isothiocyanate and integrated into a MELC library of more than 40 antibodies. These antibodies were selected for their potential to detect epitopes which may be indicative of (a) various cell types, (b) structural components of the extracellular matrix, or (c) the processes of cell proliferation, activation and adhesion. Efalizumab bound to CD11a in affected psoriatic skin by a factor 15× and 32× higher than in unaffected psoriatic skin and normal control skin, respectively. CD11a and the efalizumab binding site were primarily expressed in the extravascular dermis, whereas CD54 (ICAM-1) as its ligand was most prevalent in the dermal vessels. T lymphocytes (for which the markers were CD3, CD8, CD4, and CD45R0) were the major cellular targets of efalizumab. In contrast, NK cells were only a minor target of efalizumab. Our study demonstrated that efalizumab represents a treatment for psoriasis that primarily targets memory CD4+ and CD8+ T cells and has a high specificity for psoriatic disease activity. Moreover, we hereby introduce the novel principle of a biological drug-binding biochip assay being especially useful for the future monitoring of psoriatic skin lesions under efalizumab treatment conditions.

Topo-Proteomic in situ Analysis of Psoriatic Plaque under Efalizumab Treatment

“…Our previous investigation revealed a high abundance of efalizumab binding sites (EfaBS) in psoriatic lesions as compared to unaffected psoriatic and healthy control skin [13] . Moreover, the colocation of EfaBS with decisive epitopes of inflammatory cells, e.g.…”

“…Therefore the present study is a direct extension of our previous clinicoexperimental investigation dealing with the analysis of the inflammatory topoproteome of psoriatic skin lesions by the novel Multi-Epitope Ligand Cartography (MELC) robot technology [13] . We have recently introduced the MELC robot technology to the fields of dermatology and skin research as an innovative and highly advanced multiplex immunofluorescence microscopy allowing the mapping of the subcellular colocation of at least 100 different molecular markers, especially proteins, in one single skin tissue section [14][15][16] .…”

“…affinity reagent such as an antibody), (2) fluorescence signal imaging, and (3) soft photo-bleaching of fluorescence with subsequent point-precise computational (i) image overlay and (ii) signal colocation analysis. Meanwhile the technique has been adapted for the special methodological needs of analyzing skin tissue [13] and blood cell preparations [18] .…”

“…Moreover, the colocation of EfaBS with decisive epitopes of inflammatory cells, e.g. T cells, and skin subcompartments (dermal vessels, extravascular dermis, epidermis) was deciphered with a high resolution [13] .…”

“…Some of these patients previously underwent pretreatment MELC analysis of skin specimens [13] and a MELC toponome analysis of peripheral blood lymphocytes [18] .…”